1.3 Objetivos
1.3.2 Objetivos específicos:
• Definir o atual “estado da arte” na investigação de modificadores genéticos da idade de início (AO) na DMJ/SCA3 através de uma revisão sistemática e metanálise.
• Viabilizar a metodologia de amostragem de fenótipos extremos para a AO para a população gaúcha com DMJ/SCA3.
• Com base nos resultados da metanálise proposta, selecionar potenciais candidatos a modificadores genéticos da AO na DMJ/SCA3 e testá-los em coortes selecionadas de pacientes utilizando a metodologia de amostragem de fenótipos extremos para AO, entre outras.
• Avaliar o papel das chaperonas moleculares, em especial a DNAJB6, na modulação da AO e da velocidade de progressão neurológica da DMJ/SCA3.
39
2 Capítulo 2. O estado da arte sobre modificadores da idade de início na
DMJ/SCA3
2.1 Manuscrito 1: “Genetic risk factors for modulation of age at onset in Machado-
Joseph disease/spinocerebellar ataxia type 3: a systematic review and meta- analysis”
Artigo publicado no periódico
Journal of Neurology, Neurosurgery & Psychiatry
1 de Mattos EP, et al. J Neurol Neurosurg Psychiatry 2018;0:1–8. doi:10.1136/jnnp-2018-319200
ReseaRch papeR
Genetic risk factors for modulation of age at onset in
Machado-Joseph disease/spinocerebellar ataxia type
3: a systematic review and meta-analysis
eduardo preusser de Mattos,1,2,3 Maiara Kolbe Musskopf,2 Vanessa Leotti Torman,4,5 Maria Luiza saraiva-pereira,1,2,6,7 Laura Bannach Jardim1,2,7,8
Neurogenetics
To cite: de Mattos ep, Kolbe Musskopf M, Leotti Torman V, et al. J Neurol Neurosurg Psychiatry epub ahead of print: [please include Day Month Year]. doi:10.1136/ jnnp-2018-319200
►additional material is published online only. To view please visit the journal online (http:// dx. doi. org/ 10. 1136/ jnnp- 2018- 319200). For numbered affiliations see end of article.
Correspondence to Dr Laura Bannach Jardim, Medical Genetics service, hospital de clínicas de porto alegre, porto alegre, Rua Ramiro Barcelos 2350, Brazil; ljardim@ hcpa. edu. br Received 11 July 2018 Revised 24 august 2018 accepted 11 september 2018
© author(s) (or their employer(s)) 2018. No commercial re-use. see rights and permissions. published by BMJ.
AbsTrACT
Objectives To perform a systematic review and meta-
analysis of genetic risk factors for age at onset (aO) in spinocerebellar ataxia type 3/Machado-Joseph disease (sca3/MJD).
Methods Two authors independently reviewed reports
on the mathematical relationship between caG length at the expanded ATXN3 allele (caGexp), and other genetic variants if available, and aO. publications from January 1994 to september 2017 in english, portuguese or spanish and indexed in MeDLINe (pubMed), LILacs or eMBase were considered. Inclusion criteria were reports with >20 sca3/MJD carriers with molecular diagnosis performed by capillary electrophoresis. Non-overlapping cohorts were determined on contact with corresponding authors. a detailed analysis protocol was registered at the pROspeRO database prior to data extraction (cRD42017073071).
results eleven studies were eligible for meta-analysis,
comprising 10 individual-participant (n=2099 subjects) and two aggregated data cohorts. On average, caGexp explained 55.2% (95% cI 50.8 to 59.0; p<0.001) of aO variability. population-specific factors accounted for 8.3% of aO variance. cohorts clustered into distinct geographic groups, evidencing significantly earlier aO in non-portuguese europeans than in portuguese/ south Brazilians with similar caGexp lengths. presence of intermediate ATXN2 alleles (27–33 caG repeats) significantly correlated with earlier aO. Familial factors accounted for ~10% of aO variability. caGexp, origin, family effects and caG length at ATXN2 together explained 73.5% of aO variance.
Conclusions current evidence supports genetic
modulation of aO in sca3/MJD by caGexp, ATXN2 and family-specific and population-specific factors. Future studies should take these into account in the search for new genetic modifiers of aO, which could be of therapeutic relevance.
INTrOduCTION
Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a neurological condition characterised by expansion of a polymorphic trinu- cleotide CAG tract (CAGexp) at ATXN3. SCA3/ MJD is the most common dominantly inherited ataxia worldwide,1 2 and ATXN3 alleles with ≥45
repeats code for ataxin-3 proteins with abnormally long polyglutamine (polyQ) sequences. PolyQ-ex- panded ataxin-3 is prone to aggregation into
neuronal inclusions and exerts a gain of toxic func- tion, which leads to neuronal toxicity and degener- ation,3 similarly to what happens in Huntington’s
disease and other SCAs.4
The longer the CAGexp at ATXN3, the earlier the age at onset (AO) of disease. A large body of evidence has established that AO is not entirely explained by CAGexp, which explains 50% to 60% of the vari- ability in AO,5–8 and that AO should be modulated
by additional genetic and/or environmental factors. Several candidates have been proposed, such as apolipoprotein E genotypic status,9–11 CAG length
at normal ATXN38 12 13 and ATXN26 8 alleles, and
protein levels of the DNAJB1 chaperone.14
Most of the proposed modifiers in SCA3/MJD were not replicated or had small effects that usually improve the explanation of AO variance by not more than 1%. The greater part of the missing vari- ability in AO remains unexplained, suggesting that the main CAGexp-independent modulators of AO are still unknown. Here, we performed a system- atic review and meta-analysis of genetic risk factors associated to AO in SCA3/MJD. By analysing both aggregate and individual-participant data of more than 2000 patients from 16 countries across three continents, we were able to detect an important origin-specific effect of CAGexp on AO and to confirm the effect of some putative risk factors published previously.
MeThOds
A detailed methodology protocol for this study was registered at the PROSPERO (International Prospective Register of Systematic Reviews) data- base prior to data extraction and is available at https://www. crd. york. ac. uk/ PROSPERO/ under record CRD42017073071.
Literature search and data extraction
MEDLINE (PubMed), LILACS and EMBASE were searched from January 1994 to September 2017 for reports on genetic factors related to AO in SCA3/ MJD. Search terms employed were ‘sca3’ OR ‘mjd’ OR ‘spinocerebellar ataxia type 3’ OR ‘spinocere- bellar ataxia type-3’ OR ‘machado-joseph disease’ OR ‘machado joseph disease’ AND ‘age of onset’ OR ‘age-of-onset’ OR ‘age at onset’ OR ‘age-at- onset’. Peer-reviewed articles and meeting abstracts were included, and references were checked to guarantee maximal coverage. Two reviewers
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(EPDM and MKM) independently assessed and extracted data into evidence tables. Any disagreement regarding eligibility was discussed with a third reviewer (LBJ).
Population, exposure, comparators, outcomes, and inclusion and exclusion criteria
SCA3/MJD heterozygotes from diverse geographical origins comprised the population under study. The CAG length at CAGexp was the main exposure considered for meta-analysis; other genetic variants were included in the meta-analysis as risk factors (exposures) if reported at least twice in literature. The outcome was the quantitative variable AO defined as the age at the first symptom, usually gait ataxia. Included studies should report on both (1) molecularly confirmed SCA3/MJD symptom- atic and/or asymptomatic heterozygotes and (2) the relationship between ATXN3 CAGexp (main exposure) and AO (outcome). The term ‘carrier’ was used here as a synonym for heterozy- gotes, symptomatic or not, with one ATXN3 allele with ≥45 CAG repeats. We excluded studies reporting on <20 carriers, in languages other than English, Portuguese or Spanish. If multiple publications reported the same data, the most up-to-date and complete data set was included. Corresponding authors were contacted to check for duplicated data and to grant access to their updated, pseudonymised, individual-participant databases (IPDs), whenever possible. Otherwise, we used summary statis- tics from aggregated databases (ADs). Besides AO and CAGexp, data on gender, family, length of normal ATXN3 CAG tracts and at other CAG-containing loci and/or additional genetic variants were retrieved, if available.
risk of bias assessment and quality control
The outcome AO was poorly defined in some studies. Since most patients with SCA3/MJD develop gait ataxia as the first symptom,15 we combined in a single model carriers with known
AO of gait ataxia (AOga) or of first symptom; when both criteria were available for the same individual, AOga was chosen. Only studies that measured CAG repeats by capillary electrophoresis were considered. Participation in molecular diagnosis quality control programmes was also questioned and informed here.
Analysis and data synthesis
Boxplots were used to describe the variability on both AO and CAGexp among studies. The meta-analysis was composed of three main models. First, the global relationship between ATXN3 CAGexp length and AO was investigated using data from both IPDs and ADs,16 aiming at comparing the degree of explanation
of the variability in AO by CAGexp across studies, as reported by the linear R2 measure. A second meta-analysis used IPDs only.
Since complex models (quadratic and logarithmic) were only marginally better at explaining the data (see online supplemen- tary file 1), AO was not mathematically transformed, and linear regression was used. A third analysis tested the effects of gender, family and CAG length at the non-expanded ATXN3 allele and at other CAG-containing loci, focusing on the improvement of the R2 measure. Geographical origin and interaction between origin
and CAGexp were always included as independent variables. With the exception of ATXN1, which was considered a contin- uous variable, the effect of all CAG-containing loci was assessed as both continuous and discrete variables using CAG length groups as published previously6 8 (online supplementary file 2).
The percentage of AO variability explained by belonging to the same family was tested with a fixed-effects model. Analyses were performed using the software R V.3.4.1 with packages lsmeans
and lmSupport, and SAS OnDemand for Academics V.3.1 (SAS Institute). Graphs were generated with ggplot2. Results were considered statistically significant when p<0.05.
resuLTs
systematic review
The search yielded 641 unique abstracts (online supplemen- tary file 3); 140 studies testing the relationship between AO and CAGexp at ATXN3 were selected for the systematic review (online supplementary file 4). Thirty-one studies investigated additional modifying effects on AO, including CAG repeat length at the non-expanded ATXN3 (n=19), ATXN1 (n=5),
ATXN2 (n=6), CACNA1A (n=7), ATXN7 (n=4), HTT (n=4), TBP (n=3) and ATN1 (n=4) alleles. AO differences according
to length of GGGGCC repeats at C9ORF72, and CAG repeats at RAI1 and KCNN3 were each reported once. Another report correlated ataxin-3 and selected chaperones protein levels with AO. Allelic and/or genotypic status of single-nucleotide polymor- phisms at 15 genes were also correlated with AO, including vari- ants at ATXN3 (rs3814834, rs709930 and rs910369; n=1 each),
APOE (rs429358 and rs7412; n=4) and ATXN2 (rs7969300), BDNF (rs6265), BECN1 (rs60221525 and rs116943570), CHIP (rs6597), hCAD (rs12738235), IL1A (rs1800587), IL1B
(rs16944), IL6 (rs1800795), MT-ND3 (rs2853826), OGG1 (rs1052133), PPARGC1A (rs7665116), TNF (rs1800629) and
UCHL1 (rs5030732; n=1 each). Differences in AO according to
the degree of promoter methylation at ATXN3 were evaluated by two studies, using distinct methodologies. Gender of the affected individual and transmitting parent were correlated with AO in 14 and 6 studies, respectively. Two reports considered the effect of population of origin on AO, and one evaluated the familial dependency of AO. Data extraction, references and detailed information of all AO modifiers reported in the literature, including those not selected for meta-analysis, are described in online supplementary file 5.
After contacting all corresponding authors of studies that met the inclusion and exclusion criteria (n=11), we retrieved updated information on 10 non-overlapping IPDs and 2 ADs of symptomatic individuals only (figure 1). CAGexp and geograph- ical origin were available for all IPDs. Additional data included length of non-expanded CAG tracts at ATXN3 (n=9 cohorts), and at ATXN1, ATXN2, CACNA1A and ATXN7 (n=4 cohorts). Information on gender and family effects were available for six and seven cohorts, respectively. Geographical origin, sample sizes and retrieved data for each cohort included in the meta-analysis are summarised in table 1 and detailed in online supplementary file 6.
effect of CAGexp and geographical origin
Exposure to diverse CAGexp repeat lengths at ATXN3 was the most studied risk factor. IPD and AD retrieved from 11 studies comprised four cohorts from Europe,6 7 17 18 three from Asia,8 19 20
one from North America,6 one from Central America21 and three
from Brazil.5 22 23 Brazilian cohorts comprised the Rio Grande do
Sul (Brazil-RS) cohort23 and cohorts from other Brazilian regions
(Brazil-non-RS cohorts): namely, subjects from São Paulo State22
and those described by Neurogenetics Network, a consortium of Brazilian researchers.5 Using both IPDs and ADs, the global
linear correlation coefficient between CAGexp and log10(AO) was r=−0.743 (95% CI -0.768 to -0.713, p<0.001), meaning that, on average, the causative mutation determines about 55.2% (50.8%–59.0%) of the AO variability in SCA3/MJD worldwide.
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Neurogenetics
Figure 1 Workflow of the studies selected for the present meta-analysis.
Table 1 Studies selected for meta-analysis of the modulation of age at onset in SCA3/MJD by CAG repeat length at ATXN3 and additional factors
Cohort N, published* N, available† data type QC programme‡ Available data (risk factors) reference
Rio Grande do Sul, Brazil 463 507 IPD Yes Family, gender, origin, ATXN3, ATXN1,
ATXN2, CACNA1A, ATXN7
Souza et al23 2016
EUROSCA, Europe
403 403 IPD Yes Family, gender, origin, ATXN3, ATXN1,
ATXN2, CACNA1A, ATXN7
Tezenas du Montcel et al6 2014
Taiwan 48 347 IPD NS Gender, origin, ATXN3 Wang et al19 2012
Portugal (Mainland) 48 226 IPD Yes Origin, ATXN3 Silveira et al17 1998
China 141 141 IPD NS Family, origin,
ATXN3 (expanded allele only)
Wang et al20 2017
Netherlands 342§ 133 IPD Yes Family, origin, ATXN3 van de Warrenburg et al18 2005
São Paulo, Brazil 34 110 IPD No Origin,
ATXN3 (expanded allele only)
França Jr et al22 2009
Portugal (Azorean Islands)
93 106 IPD Yes Family, gender, origin, ATXN3, ATXN1,
ATXN2, CACNA1A, ATXN7
Raposo et al7 2015
Neurogenetics network, Brazil
481¶ 104 IPD Yes Family, gender, origin, ATXN3, ATXN1,
ATXN2, CACNA1A, ATXN7
de Castilhos et al5 2014
Cuba 22 22 IPD Yes Origin, ATXN3 González-Zaldívar et al21 2015
China 802 AD NS Origin,
ATXN3 (expanded allele only)
Chen et al8 2016
USA 110 AD NS Origin,
ATXN3 (expanded allele only)
Tezenas du Montcel et al6 2014
*Number of patients reported on the original publication.
†Number of patients whose data were obtained after contacting the corresponding author of the selected publication. ‡Participation in molecular diagnosis quality control programme.
§This study reports on Dutch and French patients, but only the Dutch cohort was available for analysis.
¶This study includes patients from the Rio Grande do Sul cohort, but only patients from other Brazilian regions were retrieved for analysis.
AD, aggregated database; EUROSCA, European Consortium on Spinocerebellar Ataxias; IPD, individual-participant database; NS, not specified; SCA3/MJD, spinocerebellar ataxia type 3/Machado-Joseph disease.
Subsequent analyses used IPD cohorts only, totaling 2099 patients. Variability of CAGexp length among the 10 cohorts (figure 2A) was wider than variability in AO (figure 2B). Inclu- sion of geographical origin increased in 8.34% the explanation of AO variability (adjusted R2=0.556; F
10,2091=263.8, p<0.001; online supplementary file 1). CAGexp significantly interacted
with origin, which improved the model by an additional 1.02% (adjusted R2=0.564; F
19,2082=144.1, p<0.001). The differential effect of CAGexp on AO among the 10 cohorts was evidenced by differences in slope and position of regression lines (figure 3A and B). Pairwise analysis of cohorts with similar slopes and/ or intercepts allowed for data aggregation into three main
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Figure 2 Variability of caG length at the expanded aTXN3 allele and age at onset in sca3/MJD. Data on caG length at the expanded ATXN3 allele and age at onset are shown for the 10 original, individual-participant data cohorts selected for meta-analysis. eUROsca, european consortium on spinocerebellar ataxias; sca3/MJD, spinocerebellar ataxia type 3/Machado-Joseph disease.
geographical/ethnic groups with differential CAGexp modula- tion of AO: an average group with heterogeneous origins (China, Cuba, Brazil-non-RS and Taiwan cohorts), the group of non-Por- tuguese Europe (EUROSCA and Netherlands cohorts) and the group with clear Portuguese origin (Azorean Islands, mainland Portugal and Brazil-RS cohorts) (figure 3C and D; online supple- mentary file 7). Table 2 presents mean AO predictions for each geographically distinct group as a function of CAGexp tracts of three different lengths.
effect of non-expanded CAG repeats at ATXN3 and other CAG-containing loci
Data on CAG length of non-expanded ATXN3, ATXN1, ATXN2,
CACNA1A and ATXN7 alleles were available from 944 patients
from four cohorts (EUROSCA, Azorean Islands, Brazil-RS and Brazil-non-RS) for meta-analysis. Inclusion of CAG length at the non-expanded ATXN3 allele did not significantly improve the correlation between CAGexp and AO (p=0.327, contin- uous variable; p=0.388, discrete variable; online supplemen- tary file 1). From the remaining candidate loci, only ATXN2 significantly improved the explanation of AO variability, with longer ATXN2 CAG tracts correlating with earlier AO (adjusted R2=0.630; F
10,933=161.6, p<0.001; online supplementary file 1). There was a significant interaction between length of the longest CAG tract at ATXN2 and CAGexp, which contrib- uted an additional 0.39% to the explanation of AO variability (p=0.020; online supplementary file 1). Presence of at least one intermediate ATXN2 allele (27–33 CAGs; 5% of alleles) significantly correlated with earlier AO (adjusted R2=0.632;
F13,930=125.7, p<0.001; figure 4A and online supplementary file 1) in individuals with CAGexp tracts of up to 73 repeats (table 3).
Family and gender effects
Information on family effects was available for 1368 patients from 565 families (online supplementary file 8). Among these, CAGexp and origin alone explained ~60% of AO variance (adjusted R2=0.599; F
11,1356=186.8; p<0.001). Inclusion of family data in a fixed-effects model increased the explana- tion by ~10% (adjusted R2=0.702; F
888,479=4.6; p<0.001; online supplementary file 1). Data on gender were available for 1468 patients, and its inclusion contributed an additional 0.3% increase in the explanation of variability in AO (adjusted R2=0.590; F
10,1457=211.9; p<0.001; online supplementary file 1). On average, male patients had younger ages at onset, espe- cially among individuals with longer CAGexp tracts (figure 4B
and online supplementary file 9). However, when considered together with CAG repeat length at ATXN2 (n=942 individ- uals), the effect of gender was not significant (p=0.08; online supplementary file 1).
Combined effects
The final and best regression model considered CAGexp, origin, family effects and ATXN2 genotypes, and explained 73.5% (95% CI 68.2 to 77.6) of the AO variance (adjusted R2=0.735;
F682,245=4.8; p<0.001; online supplementary file 1).
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Figure 3 population-specific modulation of the age at onset (aO) in spinocerebellar ataxia type 3/Machado-Joseph disease. (a) Linear regression of caGexp and aO in the 10 distinct cohorts included in the present meta-analysis (cohorts are colour-coded as in panel B). (B) same as in (a), but with individual plots for each cohort. The β coefficient of the linear regression, expressed as mean±se, is also shown for each cohort. (c) same as in (a), but with patients grouped in three main geographical origins, as a result of pairwise comparisons of intercepts and slopes of lines presented in (a). (D) same as in (c), but with geographical origins depicted separately. hexagons depict groups of patients, according to the colour scheme, and dashed lines represent linear regressions. For each plot, the R2 correlation coefficient is also shown. eUROsca, european consortium on spinocerebellar ataxias.
dIsCussION
This worldwide systematic investigation of risk factors for AO in SCA3/MJD detected that the CAGexp determines, on average, 55.2% of the phenotypic variability in AO. Additional modula- tion of AO by family factors, gender and CAG length at ATXN2 were confirmed. Interestingly, currently unknown effectors related to geographical origin were also shown to modify AO. Although several more candidates have already been proposed, data were not robust enough to be meta-analysed, and further replication studies are necessary to assess their validity as pheno- typic modulators in SCA3/MJD.
effect of CAGexp and geographical/ethnic and family background
Clear geographical/ethnic differences on the effect of CAGexp on AO tell us that a universal correlation might not applicable